Drug-resistant Infections

Treatment Options for Infections Caused by Carbapenem-resistant Gram-negative Bacteria Chris Kosmidis, 1 Garyphalia Poulakou, 2 Antonios Markogiannakis 3 and George L Daikos 4 1. Research Associate, First Department of Propaedeutic Medicine, University of Athens; 2. Consultant, Infectious Diseases, Attikon University Hospital; 3. Clinical Pharmacist, Department of Pharmacy, Laiko General Hospital; 4. Associate Professor of Medicine and Infectious Diseases, First Department of Propaedeutic Medicine, University of Athens, Athens, Greece Abstract Carbapenem-resistant Gram-negative bacteria have spread worldwide and constitute a serious public health problem. These organisms are frequently resistant to multiple classes of antimicrobials, limiting our therapeutic options. Agents with potential activity against these multi-drug-resistant bacteria include sulbactam, colistin, tigecycline, aminoglycosides, fosfomycin and, under certain conditions, β-lactams. Clinical experience on the value of each of these agents is still limited. However, in the absence of solid data, various combinations of antibiotics are frequently employed for the treatment of severe infections caused by carbapenem-resistant bacteria. Keywords Carbapenem-resistant, Acinetobacter, Pseudomonas, Enterobacteriaceae, resistance mechanisms, epidemiology, treatment, multi-drug resistance, nosocomial infections Disclosure: The authors have no conflicts of interest to declare. Received: 16 January 2012 Accepted: 1 March 2012 Citation: European Infectious Disease, 2012;6(1):28 34 Correspondence: George L Daikos, Associate Professor of Medicine and Infectious Diseases, First Department of Propaedeutic Medicine, University of Athens, Laiko General Hospital, Mikras Asias 75, Athens 115-26, Greece. E: gdaikos@med.uoa.gr Infections caused by multi-drug-resistant (MDR) bacteria constitute a major challenge for current medical practice. Carbapenems, including imipenem, meropenem, ertapenem and doripenem, are the β-lactam antibiotics with the broadest spectrum of antibacterial activity against Gram-positive, Gram-negative and anaerobic organisms. They retain reliable bactericidal activity against Gram-negative organisms producing extended-spectrum β-lactamases (ESBLs) and AmpC-type enzymes which had been major drivers of resistance in the previous decade; therefore, increased use of these agents has been established in the treatment of hospital-acquired infections. Unfortunately, Gram-negative bacteria, the most clinically relevant being Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae and other Enterobacteriaceae, are becoming increasingly non-susceptible to carbapenems, due to the acquisition of new resistance mechanisms, largely attributed to the widespread use of these antimicrobials. Carbapenem resistance is usually accompanied by resistance to all β-lactam agents and often by resistance to many other classes of antimicrobials such as quinolones and aminoglycosides, because of concurrent carriage of additional resistance determinants. Therefore, therapeutic options for these infections are extremely limited and there are no established guidelines for their management. 1 3 In this article, the authors will briefly present the epidemiology of carbapenem resistance in A. baumannii, P. aeruginosa and the Enterobacteriaceae family and discuss the available options for treatment of infections caused by carbapenem-resistant Gram-negatives. Acinetobacter baumannii A. baumannii, a Gram-negative non-fermenting bacterium, has become one of the most important nosocomial pathogens. One of its major characteristics is the ability to survive for prolonged periods in the inanimate hospital environment. It is a very successful coloniser of critically ill patients in intensive care units (ICUs) and a frequent cause of life-threatening infections, namely ventilator-associated pneumonia (VAP), bloodstream infections (BSIs), urinary tract infections (UTIs), complicated intra-abdominal infections and post-operative meningitis. 4 Acinetobacter spp. demonstrate a remarkable ability to acquire resistance determinants, illustrated by the presence in their genome of resistance islands carrying a multitude of genes that are often acquired from other bacterial species. 5 As a result, MDR Acinetobacter spp. have become a serious problem in most parts of the world, mostly affecting patients in ICUs, with resistance rates rising yearly. Moreover, treatment of infections by MDR A. baumannii has been associated with poor outcomes. 6 Carbapenem resistance is predominantly conferred by carbapenemases, such as oxacillinase (OXA)-type enzymes and metallo-β-lactamases (MBLs). 4 Of these, acquired OXA-type enzymes, belonging to families OXA-23, -24 and -58, are more prevalent. 7 Some are plasmid-mediated, explaining their rapid dissemination. OXA-type enzymes have a greater affinity for imipenem than for meropenem. MBLs (mostly of imipenemase [IMP]- and Verona integron-encoded MBL [VIM]-type and, more recently, of New Delhi MBL [NDM]-type) have also been found in A. baumannii. 8 Non-enzymatic carbapenem resistance mechanisms include membrane porin changes, such as loss of the CarO porin, and multi-drug efflux pumps. 9,10 Recently, 28 TOUCH BRIEFINGS 2012

Treatment Options for Infections Caused by Carbapenem-resistant Gram-negative Bacteria heteroresistance (subpopulations exhibiting resistance to a particular agent within a susceptible population) against meropenem has been described among apparently meropenem-susceptible A. baumannii strains. 11 Furthermore, penicillins (ampicillin/sulbactam and ticarcillin/clavulanate) and cephalosporins also seem to be affected by heteroresistance in a very recent report. 12 The clinical significance of the heteroresistance phenomenon has not been clarified. Carbapenem-resistant Acinetobacter (CRAB) is becoming prevalent in healthcare settings. In a worldwide survey (SENTRY), imipenem susceptibility rates declined progressively from 2006 to 2009 in all participating regions; for 2009, susceptibility rates for North America, Europe, Latin America and the Asia/Pacific region were 62.5 %, 45.1 %, 23.7 % and 37.4 %, respectively. 13 In another study, areas with the highest rates of resistance were the Middle East, Latin America and the Asia/Pacific region (Tigecycline evaluation and surveillance trial [TEST] study). 14 Among European countries, the highest resistance rates are observed in Greece, Italy, Spain and England. Treatment Options Agents with in vitro activity against CRAB include sulbactam, colistin, tigecycline, minocycline, rifampicin and the aminoglycosides. A. baumannii, and CRAB in particular, often causes infections in seriously ill patients with multiple comorbidities and, in this setting, the therapeutic efficacy of the aforementioned agents is not easy to assess. 15 Furthermore, resistance in Acinetobacter appears to be escalating rapidly worldwide and, more worryingly, cases of pan-resistant Acinetobacter for which there is no active agent are being increasingly reported. 16 Sulbactam Sulbactam is active against A. baumannii by inhibiting penicillin-binding protein-2 (PBP2). In most countries, this agent is only available as a co-formulation with ampicillin. Both animal studies and clinical observations have shown that sulbactam was comparable to carbapenems in the treatment of serious infections, including VAP and BSI, caused by susceptible organisms. 17,18 In most studies, a total daily dose of ampicillin/sulbactam ranging from 18 to 24 g divided into three to six doses was used. 19 However, the efficacy of sulbactam in meningitis was variable, probably because of suboptimal cerebrospinal fluid (CSF) drug levels. 20 Notably, adequate drug levels in serum are reached with a daily dose of sulbactam of 6 g for normal renal function, which corresponds to an ampicillin/sulbactam dose of 3 g every four hours. Based on these observations, sulbactam may be considered in the treatment of CRAB infections, including VAP, although data are not robust enough to recommend it for meningitis. Colistin Colistin is the agent retaining the best susceptibility profile against CRAB. Colistin showed good activity against a worldwide collection of Acinetobacter, and remained active against imipenem-resistant Acinetobacter strains as well. 13 There are, nevertheless, reports of increasing resistance to colistin among CRAB. Moreover, heteroresistance may further compromise the activity of this agent. 21,22 Clinical data have shown favourable outcomes for colistin in the treatment of MDR A. baumannii infections. Colistin was comparable to imipenem for the treatment of VAP caused by A. baumannii in a number of studies. 23,24 In view of the limited clinical data on the effectiveness of colistin for CRAB infections, combination treatment with other agents is often used. This is supported by in vitro and animal studies demonstrating synergy between colistin and various agents. 25 Most data exist on synergy with rifampicin and the carbapenems, although in vitro studies also show synergy with tetracyclines, macrolides and glycopeptides. However, few clinical data have confirmed the clinical utility of colistin combination therapy, and a retrospective study did not demonstrate a better outcome for colistin plus meropenem compared with colistin monotherapy. 26 A subsequent large retrospective study of the previous group comprised 258 patients treated over six years with colistin monotherapy or various combinations of colistin for infections by MDR Gram-negative pathogens (67.5 % A. baumannii). In this cohort, those who received colistin monotherapy or colistin/meropenem combination had a better outcome than those who received colistin in combination with other antibiotics. 27 Additionally, a higher average daily dose of colistin was associated with a lower mortality. No differences were observed in the effectiveness of colistin with regard to the specific type of Gram-negative bacterium (i.e. A. baumannii, P. aeruginosa or K. pneumoniae). In the adjusted analyses among patients with infections susceptible only to colistin, combination therapy did not offer a survival advantage. 27 Besides the intravenous formulation, alternative routes of administration of colistin have been evaluated. Although the approved indication for inhaled colistin is for the treatment of Pseudomonas spp. infections in patients with cystic fibrosis, it has also been used for the treatment of CRAB pneumonia. Inhaled colistin as adjunctive to intravenous colistin did not provide any significant benefit for patients with VAP in a case-control study, being, however, associated with faster microbiological eradication. 28,29 Other clinical studies, however, have shown promising results by using inhaled colistin concomitantly with intravenous therapy. 30,31 Intrathecal or intraventricular colistin has been used successfully for the treatment of post-operative meningitis, alone or in combination with intravenous colistin, and appears to be well tolerated. The recommended dosage is 10 mg of colistin base activity daily. 32 Tigecycline Tigecycline is a bacteriostatic agent with good in vitro activity against Acinetobacter spp. The antibiotic is not licensed for the treatment of infections by A. baumannii. Furthermore, the international microbiology societies have not implemented breakpoints for resistance against A. baumannii. 33 In a European survey, tigecycline had the lowest minimum 90 % inhibitory concentration (MIC 90 ) values among all the agents tested, and worldwide surveillance also showed good MIC 90 values (1μg/ml). 34 Additionally, tigecycline may be the only active agent in colistin-resistant CRAB. There are reports and case series of tigecycline use for CRAB VAP with favourable outcomes ranging from 20 to 84 %, although patients were frequently administered concomitant agents. 33,35 In a retrospective study of 45 patients, tigecycline monotherapy was administered to 14 patients with VAP and three with hospital-acquired pneumonia (HAP) by MDR A. baumannii, resulting in a cure rate of 76.5 % with an attributable mortality of 12 %. 36 However, in a large randomised controlled trial comparing tigecycline versus imipenem in the treatment of VAP and HAP, tigecycline failed to establish non-inferiority in the VAP arm, probably owing to pharmacokinetic (PK) data relating to critically ill patients; therefore, this agent is not recommended for the treatment of nosocomial pneumonia. 37,38 Several authors have reported resistance to tigecycline emerging during treatment for A. baumannii, EUROPEAN INFECTIOUS DISEASE 29

as well as the possibility of breakthrough infections with organisms inherently resistant to tigecycline (i.e. Proteus spp. or P. aeruginosa). 36,39 Tigecycline monotherapy should be avoided in bacteraemia, as the attained serum levels are inadequate; novel pharmacokinetic/pharmacodynamic (PK/PD) data indicate that in order to treat infections by A. baumannii, especially when targeting difficult body compartments, escalating doses of tigecycline are warranted. 38,40 Minocycline Minocycline is more active than doxycycline against A. baumannii. 41 In a survey of European Acinetobacter spp. isolates, minocycline was one of the most active agents (80.1 % susceptible). 34 Importantly, it may be the only available option for oral treatment. Unfortunately, there are no clinical data on its use for CRAB infection, but it may offer an alternative option whenever oral therapy is considered. Carbapenems Carbapenems have been used as part of combination treatment for Acinetobacter with carbapenem MICs in the non-susceptible range. In vitro data have demonstrated synergy between the carbapenems and various other agents against CRAB such as sulbactam, colistin and minocycline. 25 In addition, for the treatment of infections by A. baumannii displaying borderline carbapenem susceptibility, a higher dose (such as meropenem 2 g every eight hours) and prolonged infusion (three hours) may be used in order to improve PK/PD parameters. In an in vitro study, doripenem exhibited in vitro activity against 20.8 % of CRAB isolates (MIC 16 μg/ml for imipenem or meropenem), at a clinically achievable concentration of 4 μg/ml. 42 Doripenem possesses the advantage of stability in ambient conditions; clinical data from a randomised controlled trial of VAP with use of a double dose and prolonged infusion showed non-inferiority compared with imipenem. PK/PD applications could possibly offer additional treatment options against A. baumannii strains with borderline susceptibility to doripenem. 43 (1997 2007) showed meropenem and imipenem susceptibilities of 83 % and 79.7 %, respectively. 48 In a study from the US (2007 2009), susceptibilities to doripenem, imipenem and meropenem in 2009 were 84.1 %, 78.5 % and 87.7 %, respectively. 44 The decreased activity of imipenem compared with meropenem and doripenem is probably due to high prevalence of OprD porin loss. 44 Despite the generally low levels of resistance to the carbapenems among Pseudomonas, MDR Pseudomonas is becoming increasingly common and is a serious problem, particularly as a VAP pathogen. Treatment Options Carbapenem-resistant P. aeruginosa is predominantly an ICU pathogen, and one of the most common causes of VAP. These infections are often associated with poor outcomes. Treatment options include colistin, aminoglycosides, fluoroquinolones, fosfomycin and β-lactams. P. aeruginosa is intrinsically resistant to tigecycline. Colistin Colistin is the only agent that retains excellent activity against P. aeruginosa according to a worldwide survey, with 99.6 % of isolates testing susceptible. Reports of reduced colistin activity against P. aeruginosa have been published. 49 There is only limited experience with colistin in P. aeruginosa infections but, in a retrospective study of P. aeruginosa bacteraemia, polymyxin B was associated with increased mortality compared with β-lactams. 50 Conversely, similar clinical efficacy for colistin and imipenem was noted in a study of VAP caused by P. aeruginosa or A. baumannii, and other studies have also showed favourable outcomes with colistin. 23,27,51 Furthermore, in vitro data support the utility of colistin in combination with other agents such as carbapenems and rifampicin, but no clinical data exist. 52,53 The clinical utility of inhaled colistin for the treatment of P. aeruginosa VAP has only been evaluated in a small number of patients. 54 Intrathecal colistin has been used for treatment of Pseudomonas meningitis in combination with intravenous colistin. 55 Pseudomonas aeruginosa P. aeruginosa is implicated in several clinical syndromes, predominantly in healthcare-associated settings, such as febrile neutropenia, infections in cystic fibrosis patients, infections in intravenous drug users, UTIs, HAP, wound infections and osteomyelitis. Loss of porin, upregulation of efflux mechanisms and carbapenemases are responsible for carbapenem resistance in Pseudomonas spp. In a US survey, the most common mechanisms appeared to be efflux pump overexpression and lack of OprD porin expression. 44 AmpC overexpression was also seen, and more than one mechanism was found in the majority of isolates. Although carbapenemase production is less common as a mechanism of carbapenem resistance in P. aeruginosa, a variety of carbapenemases have been detected, including MBLs (IMP, VIM, São Paulo MBL [SPM] and German imipenemase [GIM]) and class D enzymes (OXA-198). 45 The emergence of K. pneumoniae carbapenemase (KPC)-producing P. aeruginosa was recently described from various regions. 44,46 Resistance mechanisms have differing effects on various carbapenems; OprD mutations may result in a phenotype exhibiting resistance only to imipenem, whereas efflux pump mutations affect meropenem more than doripenem or imipenem. 47 Resistance of Pseudomonas spp. to the carbapenems is less widespread than in Acinetobacter spp. An antimicrobial surveillance programme (SENTRY) of P. aeruginosa isolates spanning 10 years Beta-lactams As mentioned, resistance mechanisms exert variable effects on the activity of various carbapenems. For example, the presence of porin mutations may result in a phenotype of imipenem resistance, whereas meropenem may test susceptible. Conversely, efflux pump overexpression may be associated with a phenotype of meropenem resistance. Doripenem may display lower MICs in the presence of Pseudomonas resistance mechanisms and therefore may be a therapeutic option for imipenem or meropenem non-susceptible strains. 47 In addition, in vitro susceptibility to earlier-generation β-lactams, such as cefepime or piperacillin/tazobactam, may be retained in the presence of porin mutations. In fact, according to data from Greece, piperacillin/tazobactam retains better activity against Pseudomonas than imipenem, both in isolates from medical wards and ICUs (see www.mednet.gr/whonet). There are no outcome data for infections caused by such pathogens and it is not clear whether in vitro susceptibilities will translate into therapeutic success. Fosfomycin Promising features of fosfomycin include its bactericidal activity, good toxicity profile and lack of cross-resistance with other agents. Data on fosfomycin susceptibility are variable: in a meta-analysis, only 30.2 % of MDR Pseudomonas isolates were susceptible to fosfomycin, whereas Samonis et al. reported a susceptibility rate of 64.5 %. 56,57 However, a recent update of European Committee on Antimicrobial 30 EUROPEAN INFECTIOUS DISEASE

Treatment Options for Infections Caused by Carbapenem-resistant Gram-negative Bacteria Susceptibility Testing (EUCAST) breakpoints has removed fosfomycin from the agents with activity against P. aeruginosa, because of increasing rates of resistance. 58 In vitro synergy between fosfomycin and carbapenems or aminoglycosides has been reported. Fosfomycin-resistant mutants have been shown to emerge at a rapid rate in vitro, and emergence of resistance during therapy may be limiting the clinical utility of this agent. 59 Therefore fosfomycin should always be administered in combination with other agents, such as carbapenems, colistin or aminoglycosides. Other Agents Tobramycin and amikacin have been the aminoglycosides with most consistent activity against Pseudomonas. However, resistance phenotypes may favour gentamicin or netilmicin. Aminoglycosides are being used in combination with β-lactams against invasive P. aeruginosa infections. In the setting of carbapenem resistance, aminoglycoside monotherapy is not recommended for P. aeruginosa, especially for VAP. On the other hand, combination with colistin carries the risk of nephrotoxicity. Fluoroquinolones are an option for severe Pseudomonas spp. infection; however, carbapenem-resistant strains usually also exhibit high rates of fluoroquinolone resistance. Enterobacteriaceae Enterobacteriaceae are implicated in a wide spectrum of infections in the community and the healthcare environment, including UTIs, intra-abdominal infections, line-related infections and pneumonia. Among Enterobacteriaceae, the problem of reduced carbapenem susceptibility is most evident in K. pneumoniae, followed by Enterobacter spp. and Escherichia coli. Carbapenem resistance in Enterobacteriaceae is mediated predominantly by the production of carbapenemases, namely MBLs of IMP, VIM and NDM types, serine carbapenemases of KPC type and OXA-48. Carbapenem non-susceptibility can also be due to AmpC overexpression in conjunction with porin loss (Omp35/Omp36). VIM-type carbapenemases were first observed in K. pneumoniae in Southern Europe in the early 2000s and have since spread to other regions, such as Northern Europe and the US. In a prospective study of K. pneumoniae bacteraemia from Greece, 37.6 % of isolates carried VIM-1. 60 More recent data from that country show that the prevalence of VIM-type carbapenemases is declining as they are being replaced by KPC-type carbapenemases. 61 IMP-type carbapenemases have been described in the Far East since the 1990s. They are frequent in Japan and are also reported from China, Taiwan, Korea and Australia, but have not spread extensively to other regions of the world. NDM-type MBLs were first described in the Indian subcontinent. Their dissemination to regions such as North America, Europe and Australia has been linked to patients transferred from the Indian subcontinent, and has received considerable attention due to their extensively resistant profile. 62 64 KPC-producing K. pneumoniae was first noticed in the north-eastern parts of the US during the 2000s. Subsequently, KPC-producing Klebsiella has reached epidemic proportions in various areas such as Israel, Greece and other countries. 65 The majority of KPC-producing K. pneumoniae belongs to a single clone, ST258. Besides Klebsiella spp., KPC genes have spread to other Enterobacteriaceae. Klebsiella spp. isolates producing both KPC and VIM have been the cause of an outbreak in Greece. 66 OXA-48-producing K. pneumoniae is endemic in Turkey, but has also been described in other countries in the Middle East and elsewhere. 67 Treatment Options Carbapenems and Aztreonam Despite the carriage of carbapenemase genes, Klebsiella spp. may display low carbapenem MICs. This phenomenon has been mostly observed with VIM and IMP enzymes; in a study of 67 VIM-producing K. pneumoniae isolates, 79 % had an MIC of 4 μg/ml for at least one carbapenem. 60 Among 150 contemporary KPC-producing K. pneumoniae tested in the authors laboratory, 41 (27.3 %) had an MIC 2 μg/ml (Daikos GL, unpublished data). According to new EUCAST breakpoints (imipenem and meropenem MIC 2 μg/ml, doripenem MIC 1 μg/ml and ertapenem MIC 0.5 μg/ml), these isolates would be characterised as susceptible. This raises the question whether carbapenems can be used for infections caused by isolates producing a carbapenemase. Animal studies have shown the utility of imipenem for VIM-producing strains with MICs in the susceptible range. 68 In an in vitro PK model, meropenem 2 g every eight hours was bactericidal against KPC-producing K. pneumoniae with an MIC of 2 μg/ml. 69 In addition, doripenem has shown therapeutic potential in experimental infections caused by KPC-producing K. pneumoniae isolates with MICs up to 8 μg/ml in both immunocompetent and neutropenic mice. 70 Double carbapenem treatment with doripenem and ertapenem was tested for KPC-producing Klebsiella in vitro and in an in vivo model, taking advantage of KPC s increased affinity for ertapenem, with promising results. It was postulated that ertapenem would trap the enzyme, resulting in enhanced activity for doripenem. 71 Clinical data indicate that carbapenems may have some utility against carbapenemase-producing K. pneumoniae (CPKP). In a review of clinical cases of infections treated with carbapenem monotherapy, outcomes for CPKP infections with MIC 4 μg/ml were better than for those with an MIC of 8 μg/ml or higher, and comparable to those for infections by non-cpkp. 72 In addition, combination treatment with a carbapenem plus another active agent (aminoglycoside, colistin or tigecycline) was superior to carbapenem monotherapy and to treatment with other active agents besides carbapenems. 72 Therefore, carbapenemase-producing Enterobacteriaceae may be treated with a carbapenem, if the MIC is 4 μg/ml, using high-dose prolonged infusion regimens, preferably in combination with another active agent. Although aztreonam is not a substrate for MBLs (VIM, IMP and NDM), it has limited clinical utility as these isolates also frequently co-express ESBLs. A rabbit peritoneal abscess model showed the utility of this agent against MBL-producing Escherichia coli. 73 Thus, aztreonam could be clinically useful for infections caused by MBL-producing Enterobacteriaceae, in the absence of ESBLs. Colistin The revival of colistin was mainly associated with its use for the treatment of infections by MDR A. baumannii and P. aeruginosa. With the dissemination of carbapenemase-producing Enterobacteriaceae, colistin has emerged as an important therapeutic option. Initial reports in KPC-producing Enterobacteriaceae demonstrated good activity of colistin. 74 However, colistin resistance among KPC-producing Enterobacteriaceae has emerged: Marchaim et al. reported a 16 % colistin resistance rate among KPC-producing Klebsiella isolates, whereas Souli et al. reported a 14 % resistance rate. 65,75 The limited clinical experience with colistin monotherapy against carbapenemase-producing Enterobacteriaceae suggests that outcomes are not satisfactory. The inferior clinical efficacy of this agent as monotherapy may be associated with suboptimal dosing EUROPEAN INFECTIOUS DISEASE 31

Table 1: Recommended Regimens for the Treatment of Infections Caused by Carbapenem-resistant Gram-negative Bacteria Bacterium Regimen Acinetobacter spp. Sulbactam (usually given in combination with ampicillin) Colistin (preferably in combination with another active agent) Tigecycline (caution in VAP, bacteraemia, UTIs) Meropenem or doripenem in high-dose and prolonged infusion regimen combined with another active agent Pseudomonas Colistin preferably in combination with another active aeruginosa agent β-lactams (cefepime, piperacillin/tazobactam, if susceptible) Fosfomycin (always in combination) Aminoglycosides in combination with another agent Fluoroquinolones Enterobacteriaceae Carbapenems (if MICs 4 μg/ml; use high-dose prolonged infusion regimen in combination with another active agent, preferably colistin or gentamicin) Aztreonam, if susceptible, in combination with colistin or gentamicin Colistin in combination with another active agent Gentamicin in combination with another active agent Tigecycline (caution in VAP bacteraemia, UTIs) Fosfomycin (always in combination with other agents) UTI = urinary tract infection; VAP = ventilator-acquired pneumonia. regimens of the drug. In a retrospective study that evaluated MDR Gram-negative infections receiving several dosages of colistin, multivariate analysis of survival data showed that lower total daily dose of intravenous colistin was associated with increased mortality. 27 It is critical, therefore, to administer adequate total daily doses of colistin to critically ill patients, and particularly to those who are on renal replacement therapy, in order to attain efficacious levels according to current recommendations. 76 An additional factor that could be detrimental to patient outcomes is the delay in attaining efficacious drug concentration with the standard treatment regimen of colistin. This could be overcome by administering a loading dose of the drug. 77 As clinical outcomes with colistin monotherapy may be suboptimal, combination treatment with other active agents such as tigecycline, or carbapenems (when the MIC for the infecting organism is 4 μg/ml), may be considered. 72 In vitro data also show synergy between colistin and other agents and support this practice. 78 80 Killing kinetic studies demonstrated that the combination of imipenem with colistin exhibited improved activity against isolates susceptible either to both agents or to colistin alone. More specifically, the combination was synergistic in 50 % of the colistin-susceptible isolates and indifferent in the remaining 50 %, irrespective of the imipenem MICs. On the contrary, for isolates non-susceptible to colistin, the combination was antagonistic in 55.6 % and synergistic in only 11 % of the isolates. 79 Colistin dosing recommendations are listed in Table 1. Aminoglycosides Gentamicin is consistently the most active aminoglycoside against both VIM- and KPC-producing Enterobacteriaceae. This is due to the concomitant presence of the aac(6 )-I gene, conferring amikacin, Table 2: Suggested Colistin Dosage Colistin (CBA) Dosing Recommendation Loading dose Colistin serum concentration target x 2 x ideal body weight (kg). Example: for a target colistin serum concentration of 2 mg/l and ideal body weight of 70 kg, the loading dose must be: 2 x 2 x 70=280 mg CBA contained in 672 mg colistinmethate sodium equivalent to 8,400,000 IU Total daily Colistin serum concentration target x (1.5 x CrCL + 30). maintenance dose It is recommended that the total daily maintenance (not on renal dose is divided into two or three doses (every eight or replacement) 12 hours) and administration starts 24 hours after the loading dose. Example: for a patient with CrCL=70 ml/min and colistin serum concentration target of 2 mg/l, the total daily maintenance dose is: 2 x (1.5 x 70 + 30)=270 mg CBA contained in 648 mg colistinmethate sodium which is equivalent to 8,100,000 IU Total daily Except for haemodialysis days, for each 1 mg/l of serum maintenance dose concentration of colistin, 30 mg CBA should be (on renal administered, e.g., for a target colistin concentration of replacement 2 mg/l, the maintenance dose will be 60 mg CBA, therapy) equivalent to 1,800,000 IU (creatinine clearance is considered zero). On a haemodialysis day, for each 1 mg/l of colistin concentration in serum, 40 mg CBA should be administered after haemodialysis (30 mg as a daily maintenance dose + 30 %) Total daily For each 1 mg/l of serum concentration of colistin, maintenance dose 192 mg CBA should be administered (e.g., for a colistin (on CVVH) concentration of 2 mg/l, the daily dose will be 384 mg CBA, equivalent to 11,520,000 IU in two or three doses) Inhalation 1,000,000 2,000,000 IU/8 hours Intrathecal, 250,000 500,000 IU/24 hours intraventricular CBA = colistin base activity; CrCL = creatinine clearance; CVVH = continuous venovenous haemofiltration. 1 mg of CBA is contained in 2.4 mg colistimethate sodium which is equivalent to 30,000 international units (IU). Adapted from Garonzik et al., 2011. 76 tobramycin and netilmicin resistance, with carbapenemase genes, mostly bla VIM and bla KPC. Among 67 VIM-producing K. pneumoniae, 52.2 % were susceptible to gentamicin and 13.4 % were susceptible to amikacin. 60 In a study of 50 KPC-producing isolates, 70 % were susceptible to gentamicin, whereas only 24 % were susceptible to amikacin. 65 In contrast, NDM-producing Enterobacteriaceae typically exhibit aminoglycoside pan-resistance due to production of ribosome methylases. For serious infections, aminoglycosides may be used as combination with another active agent. In certain infections by KPC-producing Klebsiella, treatment with gentamicin may be superior to that with colistin. In a retrospective study of carbapenem-resistant Klebsiella bacteriuria, gentamicin performed better in eradication of bacteriuria than polymyxin B or tigecycline. 81 Tigecycline Tigecycline possesses enhanced in vitro activity against carbapenem-resistant Enterobacteriaceae, excluding bacteria with intrinsic resistance (i.e. Proteus spp., Morganella spp., Serratia spp.). Tigecycline was active against 46.9 % of isolates in a collection of carbapenem-resistant Enterobacteriaceae from the UK, whereas only 15.4 % of KPC-producing K. pneumoniae were susceptible in a Greek study (using EUCAST interpretative criteria). 65,82 A large controversy 32 EUROPEAN INFECTIOUS DISEASE

Treatment Options for Infections Caused by Carbapenem-resistant Gram-negative Bacteria arises from the different breakpoints for resistance that have been issued by EUCAST and the US Food and Drug Administration (FDA), the latter being higher, thus resulting in higher rates of susceptibility. 33 As a result of low maximum serum levels with usual dosing, tigecycline should be used with caution in bacteraemia, although a study showed good outcomes in cases of secondary bacteraemia. 83 In a previously mentioned Greek retrospective study, tigecycline was administered in the treatment of 23 infections by K. pneumoniae, mostly VAP and BSIs with MBL or KPC phenotype; four failures were recorded (18.2 %), whereas among 11 patients receiving monotherapy with tigecycline two failures (18.2 %) were reported. 36 Tigecycline is licensed for complicated intra-abdominal and soft-tissue infections (Europe and the US), as well as for community-acquired pneumonia (only in the US). PK data, however, suggest that the drug cannot be recommended for UTIs and VAP. Large randomised controlled trials in the treatment of MDR Enterobacteriaceae are currently lacking. Nevertheless, given the spread of NDM- and KPC-producing Enterobacteriaceae, the vast majority of off-label use of tigecycline is administration as part of the treatment for such pathogens. Fosfomycin Fosfomycin has variable in vitro activity against carbapenemaseproducing Enterobacteriaceae. Among 79 carbapenemase-producing Enterobacteriaceae, 94.9 % were susceptible to fosfomycin. 84 In another study, 54 % of KPC-producing Klebsiella isolates were susceptible to fosfomycin. 65 Fosfomycin may be the only active agent against KPC-producing Klebsiella. Synergy has been demonstrated between fosfomycin and the carbapenems. 85,86 Unfortunately, very limited data exist on the clinical outcome of infections by carbapenem-resistant Enterobacteriaceae treated with fosfomycin; in a series of 11 ICU patients with carbapenem-resistant Klebsiella, fosfomycin was used in combination with other antibacterials with good outcomes. 87 As mentioned before, the rapid emergence of resistance precludes use of this agent as monotherapy, except in the treatment of uncomplicated lower UTIs. Newer Agents in Development Several novel agents offer promise in the treatment of infections by MDR Gram-negative bacilli and are under various stages of development. Plazomicin (formerly ACHN-490) is a derivative of sisomicin not inactivated by the usual aminoglycoside-modifying enzymes, with activity against Enterobacteriaceae, Acinetobacter and Pseudomonas. 88,89 However, it cannot be used against bacteria producing 16S ribosomal RNA (rrna) methylases, which are frequently co-produced with NDM-1 carbapenemases. Avibactam (NXL-104) is a carbapenemase inhibitor with activity against serine carbapenemases such as KPC, but not against MBLs. 90 Combinations of avibactam with various β-lactams have shown promising results. BAL30376 is a monobactam clavulanic acid combination with activity against MBLs. 91 Closing Remarks In concluding this brief article, the authors have to underline once more that the data presented herein are not based on well-designed clinical studies. Given the paucity of optimal treatment for carbapenemase-producing micro-organisms, a wise exploitation of the PK/PD of available agents has to be a priority. 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